1. Field of the Invention
The present invention relates generally to the in vitro production of polysaccharides, cellooligosaccharides and more particularly to the synthetic production of a novel form of cellulose I.
3. Description of the Related Art
Cellulose is the most abundant macromolecule on earth (Brown, 1985). It serves a major structural role in the cell wall of plants, some algae, and certain fungi and is the primary component of economically important products such as wood, cotton, and paper. Because of its tremendous abundance and its physiological and economic importance, many attempts at in vitro cellulose synthesis have been made with cell-free systems from various sources during the past three decades (Delmer, 1987; Brown, 1989b; Read and Delmer, 1991). The greatest progress has been made using Acetobacter xylinum as an experimental model (Ross et al., 1987; Brown, 1989a; Lin and Brown, 1989; Lin et al., 1990; Saxena et al., 1990; Wong et al., 1990; Mayer et al., 1991; Saxena et al., 1991). However, until very recently (Okuda et al, 1993) no preparation from higher plant cells has ever been shown to be capable of synthesizing true microfibrillar cellulose or even appreciable quantities of .beta.-1,4-glucan (Read and Delmer, 1991). In the present inventors previous work a considerable progress was made over earlier research of in vitro cellulose synthesis (Okuda et al, 1993) . Although not yet approaching cellulose synthesis rates in vivo the present inventors have clearly detected a cellulose product synthesized in vitro.
The methods for synthesizing cellulose in vitro are as follows. The plasma membrane from cotton fibers is extracted with 50 mM Tris-HCl buffer (pH 7.5), containing 5 nM EGTA, 20% PEG and combination of protease inhibitors. The resulting plasma membrane fraction is solubilized in 10 nM Tris-HCl (pH 7,4), containing 1% digitonin, 1 mM EDTA, 1 mM EGTA, 10% glycerol. The in vitro product is synthesized under conditions favoring .beta.-1,4-glucan synthesis. The resulting in vitro product is characterized by solubility, enzymatic digestion, degree of polymerization (DP) determination, methylation linkage analysis, x-ray diffraction and transmission electron microscopy (TEM) coupled with autoradiography and CBHI-gold labeling. Although the product synthesized by this method is cellulose, it is cellulose II. Thus, until the present invention described herein, there has been no method known to synthesize the cellulose I polymorph in vitro from higher plant extracts.
Native cellulose predominantly occurs as a fibrillar crystalline allomorph designated cellulose I. Cellulose I consists of a crystalline array of glucan chains, all of which are oriented parallel to one another (Preston, 1974). Cellulose I can be transformed to the more stable allomorph, cellulose II, via chemical treatments that alter the crystal structure (R.ang.nby, 1952; Sarko, 1976). This change is strictly irreversible, and no process has been known that gives cellulose I either by recrystallization or by polymerization in vitro. Cellulose II recently has been found to have a folded chain, antiparallel conformation (Kuga, et al, 1993). It has been argued that cellulose I is thermodynamically metastable and, therefore, that living organisms control crystallization in a manner not duplicated under acellular conditions (R.ang.nby, 1952; Sarko, 1976; Blackwell, 1982; Sawyer and George, 1982).
Native celluloses exist in a polymorphic form known as cellulose I or crystalline polymorphs thereof (Preston, 1974). This polymorph is known to contain glucan chains that are parallel to each other. The cellulose I polymorph is characteristic of almost all native celluloses synthesized by living systems to date. Cellulose II is only rarely synthesized under natural conditions. Halicystis (an alga) (R.ang.nby, 1952; Sisson, 1938; Roberts, 1991), Sarcina (a bacterium) (Roberts, 1991; Roelfsen, 1959) and a mutant strain of Acetobacter xylinum (a bacterium) that produces folded-chain cellulose II (Kuga et al., 1993) are the few known organisms producing native cellulose II. Cellulose I is a metastable polymorph; it exists in a more thermodynamically unstable form than that of the cellulose II polymorph. In this form, the glucan chains are antiparallel to one another, and there is one additional hydrogen bond linking each glucose residue. Cellulose II is formed when cellulose I is dissolved and reprecipitated.
Electron diffraction analysis has shown that native celluloses are synthesized in a crystalline form. The electron diffraction patterns are characteristic for each type of cellulose polymorph or allomorph (both words are used interchangeably in this invention). That is, cellulose I is a polymorph that has characteristic electron diffraction patterns different from those of cellulose II. At the ultrastructural level, the crystalline form of cellulose can be visualized by a variety of electron microscope techniques, among them negative staining. This technique is useful in that it can identify small aggregates of glucan chains down to a mean diameter of approximately 12-15 .ANG..
In vitro synthesis of cellulose has been one of the most difficult, yet important and challenging topics of the early stages of macromolecular science. Much effort has been devoted to regio- and stereoselective preparations of cellulose, i.e., construction of stereoregular polysaccharides having .beta.-1,4 glycosidic linkages. The chemical approaches so far attempted, however, have failed to solve the problem in spite of the remarkable development of modern synthetic methods (Klar, 1963; Husemann, et al., 1966; Hirano, 1973; Schuerch, 1972; Uryu, et al., 1985).
In one attempt, the condensation of 2,3,6-glucose tricarbanilate with phosphorus pentoxide in a mixture of chloroform/dimethyl sulfoxide gave branched products, however, the molecular weight of the resulting polysaccharide after removing the protecting group was low (Husemann, 1966). In a Lewis acid catalyzed reaction, 1,4-Anhydro-2,3,6-tri-O-benzyl-a-D-glucose has been polymerized giving rise to polymers having mixed structures of .beta.-1,4 (cellulose-type) and .alpha.-1,4 (amylose-type) linkages. Uryu and coworkers investigated the possibility of synthesizing polysaccharides having .beta.-1,4 linkages (cellulose) by the cationic ring-opening polymerization of 1,4-anhydroglucose derivatives. However, stereoregular polysaccharides having the desired structure were not obtained due to the lack of regioselective ring opening (Uryu, et al., 1985).
Concerning a stepwise synthesis of cellooligosaccharide derivatives, several oligomers up to an octamer have been synthesized starting from allyl 2,3,6-tri-O-benzyl-4-O-(p-methoxybenzyl)-.beta.-D-glucoside; however, elimination of the protecting groups from a specific oligomer, e.g., cellooctaose, has not been achieved (Nakatsubo, 1989). The in vitro synthesis of cellulose utilizing biosynthetic pathways has been reported exploiting Acetobacter xylinum (Colvin, 1959) or Phaseolus aureus extracts (Elbein, 1964) with a nucleoside diphosphate sugar (ADP, CDP, or GDP-glucose) as the substrate.
Two of the present inventors have reported a different approach for the synthesis of cellulose, i.e., by a transglycosylation reaction (condensation polymerization) catalyzed by cellulase, an extracellular cellulose hydrolytic enzyme, with .beta.-D-cellobiosyl fluoride as a glycosyl donor (Kobayashi, et al., 1991). .beta.-D-cellobiosyl fluoride was chosen as the activated glycosyl donor because a disaccharide is the smallest molecule recognized by the enzyme. The configuration of the C1 fluorine atom of the starting material was designed to form a reactive intermediate leading to a .beta.-1,4 product (cellulose) via a "double displacement mechanism" (Lai, 1974) at the active site of the enzyme. The major advantage of this approach was that it did not involve protection and deprotection of the hydroxyl groups. Several organic solvents/buffers were investigated with an acetonitrile/acetate buffer being the most preferred, and a 5:1 ratio was found to be the best ratio for the production of cellulose. Although cellulose was produced by this method, the product was cellulose II. So, unfortunately, there was still no in vitro method to synthesize the more useful cellulose I polymorph. In European Patent Specification application No. 87307891.9 (Brown et al., 1987) in vitro cellulose synthesis was reported. However such cellulose was not synthesized but created only after washing a synthesized product with strong base or other solubilizing agents to remove contaminants.
The present invention overcomes these and other drawbacks inherent in the prior art by providing a method of producing cellulose I either synthetically by a non-biological reaction, or by using a biological catalyst in vitro.